This invention relates to recombinant DNA technology, nucleic acids, vectors and methods for use in a recombinational cloning or subcloning, and more specifically for constructing expression vectors by using recombination proteins in vitro or in vivo through site-specific recombination.
Recombinant DNA technology, also called gene cloning or molecular cloning, is widely used to transfer genetic information, i.e. DNA, from one organism to another. A typical recombinant DNA experiment often follows the following procedure. First, the DNA (e.g., the cloned DNA, insert DNA, target DNA, or foreign DNA) from a donor organism is extracted, enzymatically cleaved (or cut/digested), and joined (ligated) to another DNA entity (e.g. a cloning vector) to form a new, recombinant DNA molecule (or cloning vector-insert DNA construct). Second, this cloning vector-insert DNA construct is transferred into and maintained within a host cell, such as transformation of a bacterial host cell by the construct. Third, those host cells that take up the DNA construct (transformed cells) are identified and selected from those that do not. In addition, if required, a DNA construct can be prepared to ensure that the protein product that is encoded by the cloned DNA sequence is produced by the host cell.
Accordingly, this traditional cloning methods using restriction enzymes and ligase can be time consuming, especially when a specific expression vector is required for transferring the target gene into a heterologous host cell, such as a mammalian cell. The specific expression vector may not contain matching restriction sites for the donor DNA. Extensive reengineering of the expression vector may be required to introduce the matching restriction sites into the vector so that the vector and the insert DNA can be ligated to produce the final construct. Alternatively, multiple restriction enzymes may have to be employed to generate an insert DNA having suitable restriction sites for ligation with the vector. In this case, reaction conditions for each restriction enzyme may differ such that it is often necessary to perform a few separate restriction digestion reactions to obtain the desired insert. Further, the efficiency of direct ligation between the vector and insert may be very low, especially between large fragments. As a result, the whole procedure is tedious, and the final yield of the correctly ligated construct can be low.
Site-specific recombination represents another useful method of recombinant DNA technology. This method employs a site-specific recombinase, an enzyme which catalyzes the exchange of DNA segments at specific recombination sites. Site-specific recombinases present in some viruses and bacteria, and have been characterized to have both endonuclease and ligase properties. These recombinases, along with associated proteins in some cases, recognize specific sequences of bases in DNA and exchange the DNA segments flanking those segments. Landy, A. (1993) Current Opinion in Biotechnology 3:699-707.
A typical site-specific recombinase is Cre recombinase. Cre is a 38-kDa product of the cre (cyclization recombination) gene of bacteriophage P1 and is a site-specific DNA recombinase of the Int family. Sternberg, N. et al. (1986) J. Mol. Biol. 187: 197-212. Cre recognizes a 34-bp site on the P1 genome called loxP (locus of X-over of P1) and efficiently catalyzes reciprocal conservative DNA recombination between pairs of loxP sites. The loxP site consists of two 13-bp inverted repeats flanking an 8-bp nonpalindromic core region. Cre-mediated recombination between two directly repeated loxP sites results in excision of DNA between them as a covalently closed circle. Cre-mediated recombination between pairs of loxP sites in inverted orientation will result in inversion of the intervening DNA rather than excision. Breaking and joining of DNA is confined to discrete positions within the core region and proceeds on strand at a time by way of transient phophotyrosine DNA-protein linkage with the enzyme. Other examples of site-specific recombination systems include the integrase/att system form bacteriophage xcex and the FLP/FRT system from the Saccharomyces cerevisiae 2pi circle plasmid.
These site-specific recombination systems have been used in vivo to facilitate recombination between different vectors. Waterhouse et al. used an in vivo method to join light and heavy chains of an antibody. The light and heavy chains were cloned in different phage vectors between loxP and loxP 511 sites that were used to transform new E. coli cells. Waterhouse, P. et al. (1993) Nucleic Acid Res. 21:2265-2266. Cre acted on two parental molecules, one plasmid and another phage, in the host cells to produce four products in equilibrium: two different cointegrates (produced by recombination at either loxP or loxP511 sites), and two daughter molecules, one of which was the desired product. Schlake and Bode used an in vivo method to exchange expression cassettes at defined chromosomal locations, each flanked by a wild type and spacer-mutated FRT recombination site. Schlake and Bode (1994) Biochemistry 33:12746-12751. A double-reciprocal crossover was mediated in cultured mammalian cells by using the FLP/FRT system for site-specific recombination. Aoki et al. used a shuttle plasmid (pAdMCS) that carried a gene of interest, a loxP site, the adenoviral 5-LTR and packaging signal 0 to 1 mu, and a multiple cloning site. Aoki et al. (1999) Mol. Med. 5:224-231. The shuttle plasmid was linearized by a restriction enzyme Nhel and recombined with Clal-digested adenoviral cosmid in vitro. Cre recombinase produced the full-length recombinant adenoviral vector in vitro by an exchange of region distal to the loxP site linearized in these two molecules.
The present invention relates to compositions, kits, and methods for use in a recombinational cloning or subcloning. In particular, the present invention provides novel methods for constructing expression vectors by using site-specific recombinases in vitro. These method may be used for high throughput screening of genes, functional genomics and other human genome projects.
In one aspect, the present invention provides a double-stranded circular donor DNA for transferring a donor DNA sequence into expression vectors. The circular donor DNA comprises: a donor DNA sequence; a donor recombination site; at least one selectable marker, the circular donor DNA not including an origin of replication.
The donor DNA sequence may be any gene of interest or any synthetic DNA sequence which is needed to be transferred into an expression vector. For example the donor DNA segment may be a sequence derived from cDNA of a particular gene or one of the members of a cDNA library. The donor DNA may also be a genomic DNA that contains the coding region interrupted with non-coding sequences.
In another aspect, the present invention also provides a library of double-stranded circular donor DNAs that may be used for high throughput screening. The library of double-stranded circular DNA comprises: a donor DNA sequence which varies within a library of donor DNA sequences; a donor recombination site; and at least one selectable marker, the circular donor DNA not including an origin of replication.
The library of donor DNA sequences may be a library of cDNA or genomic DNA derived from any desirable sources. For example, the library of donor DNA sequences may be a cDNA library from single human chromosomes.
The circular donor DNA may further comprise a promoter sequence that controls expression of the donor DNA sequence. The promoter may be any array of DNA sequences that interact specifically with cellular transcription factors to regulate transcription of the downstream gene. The promoter may be derived from any organism, such as bacteria, yeast, insect and mammalian cells and viruses. Examples of the promoter include, but are not limited to, E. coli lac and trp operons, the tac promoter, the bacteriophage xcexxcex2L promoter, bacteriophage T7 and SP6 promoters, xcex2-actin promoter, insulin promoter, human cytomegalovirus (CMV) promoter, HIV-LTR (HIV-long terminal repeat), Rous sarcoma virus RSV-LTR, simian virus SV40 promoter, baculoviral polyhedrin and p10 promoter.
The promoter may also be an inducible promoter that regulates the expression of downstream gene in a controlled manner. Examples of inducible promoters include, but are not limited to, the bacterial dual promoter (activator/repressor expression system) which regulates gene expression in mammalian cells under the control of tetracycline and its analogs and promoters that regulate gene expression under the control of factors such as heat shocks, steroid hormones, heavy metals, phorbol ester, the adenovirus E1A element, interferon, or serum.
The donor recombination site may be any segment or arrays of DNA sequence recognized by a site-specific recombinase which catalyzes site-specific fusion between the circular donor DNA and an acceptor vector. The site-specific recombinase may be a recombinase, a transposase or an integrases.
In one variation, the recombination site is a lox site that is recognized by the Cre recombinase of bacteriophage PI. Example of lox site includes, but are not limited to, loxB, loxL, loxR, loxP [SEQ ID NO:1], loxP3, loxP23, loxxcex9486, loxxcex94117, loxP511 [SEQ ID NO:2], and loxC2 [SEQ ID NO:3].
In another variation, the recombination site is a recombination site that is recognized by a recombinases other than Cre. Examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage xcex (e.g. att1, att2, att3, attP, attB, attL, and attR), the FRT sites recognized by FLP recombinase of the 2pi plasmid of Saccharomyces cerevisiae, the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thruingiensis. 
The example of site-specific recombinase include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage xcex, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, Tpnl and the xcex2-lactamase transposons, and the immunoglobulin recombinases,
The selectable marker of the circular donor DNA may be any functional element for facilitating subsequent identification and selection of clones of the recombination product under suitable conditions. The selectable marker may encode any functional element, such as protein, peptide, RNA, binding site for RNA and proteins, or products that provide resistance to organic or inorganic agents. Examples of selectable markers include, but are not limited to, reporter genes such as -galactosidase (GAL), fluorescent proteins (e.g., GFP, GFP-UV, EFFP, BFP, EBFP, ECFP, EYFP), secreted form of human placental alkaline phosphatase (SEAP), xcex2-glucuronidase (GUS)); resistance genes against antibiotics (e.g. neomycin (G418) or hygromycin resistant gene, puromycin resistant gene), yeast seletable markers leu2-d and URA3, apoptosis resistant genes (e.g. baculoviral p35 gene), and antisenoligonucleotides.
The circular donor DNA may optionally include an affinity tag for selection and isolation of protein product encoded by the donor DNA segment. Examples of such an affinity tag include, but are not limited to, a polyhistidine tract, polyarginine, glutathione-S-transferase (GST), maltose binding protein (MBP), a portion of staphylococcal protein A (SPA), and various immunoaffinity tags (e.g. protein A) and epitope tags such as those recognized by the EE (Glu-Glu) antipeptide antibodies. The affinity tag may be positioned at either the amino- or carboxy-terminus of the donor DNA.
The present invention also provides a circular acceptor vector for generating recombinant expression vector. The vector comprises an origin of replication; and an acceptor recombination site capable of recombining with a donor DNA. Optionally, the acceptor vector may not include a promoter for regulating expression of the donor DNA.
The circular acceptor vector may be any vector that can transform, transfect or transduce a host cell. The acceptor vector may be a plasmid, a phage or a viral vector as long as it is able to replicate in vitro or in a host cell, or to convey the donor DNA to a desired location within a host cell. Examples of host cells include, but are not limited to, bacterial (e.g. E. coli, Bacillus subtilis, etc.), yeast, animal, plant, and insect cells.
In one variation, the circular acceptor vector may be a prokaryotic plasmid. Optionally, the acceptor vector may comprise a prokaryotic termination sequence. Examples of the prokaryotic termination sequence include, but are not limited to, the T7 termination sequence, the TINT, TL1, TL2, TL3, TR1, TR2, T6S termination signals derived from the bacteriophage xcex.
In another variation, the circular acceptor vector may be a mammalian expression vector. The mammalian expression vector contains one or more eukaryotic marker genes, appropriate eukaryotic transcriptional and translational termination signals and a sequence that signals polyadenylation of the transcript messenger RNA (mRNA), and an origin of replication that functions in a mammalian host cell. Examples of the eukaryotic polyadenylation sequence include, but are not limited to, the Herpes simplex virus thymidine kinase polyadenylation sequence, the bovine growth hormone polyadenylation sequence, and the simian virus 40 polyadenylation sequence.
Optionally, the eukaryotic expression vector may also carry an origin of replication and selectable marker genes that function in bacterial cells, forming a shuttle vector.
In yet another variation the circular acceptor includes a promoter for regulating expression of the donor DNA sequence carried by a circular donor DNA of the invention. According to this variation, the recombination site may be placed downstream of the promoter and the transcription initiation site in the acceptor vector.
In yet another variation, the circular acceptor may be a yeast expression vector such as a S. cerevisiae expression vector. Various types of S. cerevisiae expression vector include, but are not limited to, episomal or plasmid vector, integrating vectors, and yeast chromosomes (YACs).
In yet another variation, the circular acceptor vector may be a baculovirus DNA, such as wild type or mutant genomes of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV).
Optionally, a baculoviral acceptor vector according to the present invention may not contain a polyhedrin promoter. Instead, the polyhedrin or the baculoviral p10 promoter can be positioned upstream of the donor DNA sequence of the circular donor DNA of the present invention.
The present invention also provides kits for generating recombinant vectors. In one embodiment, the kit comprises: a double-stranded circular donor DNA comprising a donor DNA sequence, a donor recombination site, and at least one selectable marker, the circular donor DNA not including an origin of replication; and a circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.
In another embodiment, the kit comprises: a library of double-stranded circular donor DNA comprising a donor DNA sequence which varies within a library of donor DNA sequences, a donor recombination site, and at least one selectable marker, the circular donor DNA not including an origin of replication; and a circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.
In yet another embodiment, the kit comprises: one or more linear donor DNA comprising a donor DNA sequence; a linear driver DNA comprising a promoter sequence, a recombination site, and at least one selectable marker, ligation of the linear donor DNA and the linear driver DNA resulting in a circular donor DNA; and a circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.
The present invention also provides a method for generating recombinant expression vector in vitro through site-specific recombination between a circular donor DNA and circular acceptor DNA, each containing recombination site recognized by the recombinase. The method comprises: contacting a circular double-stranded donor DNA and a circular acceptor vector in the presence of a recombinase under conditions suitable for the circular double-stranded donor DNA and circular acceptor vector to recombine to form a single fused circular vector. In this method, the circular double-stranded donor DNA comprises a donor DNA sequence, a donor recombination site, and at least one selectable marker, but not including an origin of replication. The circular acceptor vector comprises an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA. The promoter for regulating expression of the donor DNA may be contained in either the donor DNA or acceptor vector.
According to this method, the circular donor DNA containing a site-specific recombination site may be recombined with a circular acceptor vector in the presence of Cre recombinase. The recombination sites on the circular donor DNA and the circular acceptor vector may each contain a lox site.
The method may further include steps of transforming, transfecting or transducing a host cell and selecting the correctly fused recombinant vector based on the selectable phenotype conferred by the selectable marker gene on the recombinant vector.
The present invention also provides a method for generating recombinant expression vectors from linear DNA segments in vitro. The method comprises: ligating one or more double-stranded linear donor DNA which includes a donor DNA sequence with a double-stranded linear driver DNA which includes a promoter sequence and a donor recombination site to form a single circular donor DNA, the singular circular donor DNA not including an origin of replication, where the donor DNA sequence is under the transcriptional control of the promoter; and contacting the circular donor DNA and a circular acceptor acceptor vector in the presence of a recombinase to form a single fused circular vector. In this method, the circular acceptor vector comprises an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.
According to this method, the linear donor DNA and linear driver DNA may contain matching restriction sites or other type of annealing sites so as to be ligated to form a circulaized DNA. The linear donor and driver DNAs may be derived from PCR amplification products.
The present invention also provides a method for high throughput production of recombinant expression vectors from linear DNA segments in vitro. The method comprises: ligating a library of double-stranded linear donor DNAs, where each member of the library includes a donor DNA sequence, with a double-stranded linear driver DNA which includes a promoter sequence and a donor recombination site to form a single circular donor DNA, the singular circular donor DNA not including an origin of replication, where the donor DNA sequence is under the transcriptional control of the promoter; and contacting the circular donor DNA and a circular acceptor acceptor vector in the presence of a recombinase to form a single fused circular vector. In this method, the circular acceptor vector comprises an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.
According to this method, the library of double-stranded linear donor DNAs may be DNAs amplified from a library of cDNA clones. The library of cDNA clones may be arrayed in a multi-well plate such as 96- and 384-well plates. The library of cDNA clones may be a cosmid or phage library.
Also according to the method, ligating the library of double-stranded linear donor DNAs with a double-stranded linear driver DNA may be performed by Ligation Independent Cloning (LIC). Alternatively, ligating the library of double-stranded linear donor DNAs with a double-stranded linear driver DNA may be performed in the presence of T4 DNA ligase.
The method may further include a step of transferring the recombinant expression vector into a host and isolating the protein expressed from the vector by affinity tagging. The affinity tagging may be based on a polyhistidine tag (e.g. His6), a protein tag (e.g., GST, maltose binding protein) or an epitope tag (e.g. an EE ag).
The methods of the present invention allow rapid and efficient generation of expression vectors containing the gene of interest without bacterial cloning. Direct ligation of linear donor DNA and linear driver DNA to generate a circular donor DNA allows for efficient cloning of donor DNA such as a cDNA library into an expression vector in an automated and high throughput manner. The methods can be used in a wide variety of high throughput arrays for functional genomics, protein genomics (proteomics), and other human genome projects.